Inhibitors of type 2 vascular endothelial growth factor receptors
The present disclosure relates to novel vascular endothelial growth factor receptor (VEGFR)-binding polypeptides and methods for using these polypeptides to inhibit biological activities mediated by vascular endothelial growth factors (VEGFs). The present disclosure also provides various improvements relating to single domain binding polypeptides.
1. A polypeptide comprising a modified fibronectin type III tenth ( 10 Fn3) domain, wherein the modified 10 Fn3 domain (i) comprises a modified amino acid sequence in one or more of the AB, BC, CD, DE, EF and FG loops relative to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5, (ii) comprises an amino acid sequence that is at least 60% identical to SEQ ID NO: 5, (iii) binds to a target molecule not bound by the wild-type 10 Fn3 domain, (iv) comprises a C-terminal tail comprising the amino acid sequence EIDKPSQ (residues 88-94 of SEQ ID NO: 184) or EIDKPCQ (residues 88-94 of SEQ ID NO: 194); and (v) comprises a moiety that binds to a different target, a labeling moiety, a moiety that facilitates protein purification, or a moiety that provides improved pharmacokinetics.
2. The polypeptide of claim 1 , wherein the moiety that provides improved pharmacokinetics reduces the clearance rate of the polypeptide in a mammal relative to the unmodified polypeptide.
3. The polypeptide of claim 2 , wherein the moiety is selected from the group consisting of polyethylene glycol, a sugar, an Fc fragment and serum albumin.
4. The polypeptide of claim 3 , wherein the moiety is a polyethylene glycol moiety.
5. The polypeptide of claim 1 , wherein the moiety is a labeling moiety.
6. The polypeptide of claim 5 , wherein the labeling moiety is selected from the group consisting of a radioactive moiety, a fluorescent moiety, a chromogenic moiety, a chemiluminescent moiety, and a hapten moiety.
7. The polypeptide of claim 1 , wherein the moiety that facilitates protein purification is a protein tag selected from the group consisting of a histidine tag, a FLAG tag, a myc tag and a GST tag.
8. The polypeptide of claim 1 , wherein the 10 Fn3 domain is at least 65% identical to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5.
9. The polypeptide of claim 1 , wherein the 10 Fn3 domain is at least 70% identical to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5.
10. The polypeptide of claim 1 , wherein the 10 Fn3 domain is at least 75% identical to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5.
11. The polypeptide of claim 1 , wherein the 10 Fn3 domain is at least 80% identical to the wild-type 10 Fn3 domain set forth in SEQ ID NO:5.
12. An expression vector comprising a nucleic acid encoding a polypeptide comprising a modified fibronectin type III tenth ( 10 Fn3) domain, where the modified 10 Fn3 domain (i) comprises a modified amino acid sequence in one or more of the AB, BC, CD, DE, EF and FG loops relative to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5 , (ii) comprises an amino acid sequence that is at least 60% identical to SEQ ID NO: 5, (iii) binds to a target molecule not bound by the wild-type 10 Fn3 domain, and (iv) comprises a C-terminal tail comprising the amino acid sequence EIDKPSQ (residues 88-94 of SEQ ID NO: 184) or EIDKPCQ (residues 88-94 of SEQ ID NO: 194).
13. The expression vector of claim 12 , wherein the 10 Fn3 domain is at least 65% identical to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5.
14. The expression vector of claim 12 , wherein the 10 Fn3 domain is at least 70% identical to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5.
15. The expression vector of claim 12 , wherein the 10 Fn3 domain is at least 75% identical to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5.
16. The expression vector of claim 12 , wherein the 10 Fn3 domain is at least 80% identical to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5.
17. An isolated host cell comprising a nucleic acid encoding a polypeptide comprising a modified fibronectin type III tenth ( 10 Fn3) domain, wherein the modified 10 Fn3 domain (i) comprises a modified amino acid sequence in one or more of the AB, BC, CD, DE, EF and FG loops relative to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5, (ii) comprises an amino acid sequence that is at least 60% identical to SEQ ID NO: 5, (iii) binds to a target molecule not bound by the wild-type 10 Fn3 domain, and (iv) comprises a C-terminal tail comprising the amino acid sequence EIDKPSQ (residues 88-94 of SEQ ID NO: 184) or EIDKPCQ (residues 88-94).
18. A method of producing the polypeptide comprising a modified fibronectin type III tenth ( 10 Fn3) domain, wherein the modified 10 Fn3 domain (i) comprises a modified amino acid sequence in one or more of the AB, BC, CD, DE, EF and FG loops relative to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5, (ii) comprises an amino acid sequence that is at least 60% identical to SEQ ID NO: 5, (iii) binds to a target molecule not bound by the wild-type 10 Fn3 domain, and (iv) comprises a C-terminal tail comprising the amino acid sequence EIDKPSQ (residues 88-94of SEQ ID NO: 184) or EIDKPCQ (residues 88-94 of SEQ ID NO: 194),the method comprising culturing the host cell of claim 17 to express the polypeptide and purifying the polypeptide.
19. The method of claim 18 , wherein the 10 Fn3 domain is at least 65% identical to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5.
20. The method of claim 18 , wherein the 10 Fn3 domain is at least 70% identical to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5.
21. The method of claim 18 , wherein the 10 Fn3 domain is at least 75% identical to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5.
22. The method of claim 18 , wherein the 10 Fn3 domain is at least 80% identical to the wild-type 10 Fn3 domain set forth in SEQ ID NO: 5.